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Thermal Cycling Profile for Standard PCR

Initial denaturation It is very important to denature the template DNA completely. Initial heating of the PCR mixture for 2 minutes at 9495C is enough to completely denature complex genomic DNA so that the primers can anneal to the template as the reaction mix is cooled. If the template DNA is only partially denatured, it will tend to snap-back very quickly, preventing efficient primer annealing and extension, or leading to self-priming, which can lead to false-positive results. Denaturation step during cycling

Denaturation at 9495C for 2030 seconds is usually sufficient, but this must be adapted for the thermal cycler and tubes being used. (For example, longer times are required for denaturation in 500 l tubes than in 200 l tubes.) If the denaturation temperature is too low, the incompletely melted DNA snaps-back as described earlier, thus giving no access to the primers. Use a longer denaturation time or higher denaturing temperature for GC-rich template DNA.

Note: Never use a longer denaturation time than absolutely required for complete denaturation of template DNA. Unnecessarily long denaturation times decrease the activity of Taq DNA Polymerase.

Primer annealing For most purposes, annealing temperature has to be optimized empirically. The choice of the primer annealing temperature is probably the most critical factor in designing a high specificity PCR. If the temperature is too high, no annealing occurs, but if it is too low, non-specific annealing will increase dramatically. Primer-dimers will form if the primers have one or more complementary bases
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